Immunohistochemical localization of carbonic anhydrase IV in the human parotid gland

Abstract

Carbonic anhydrases (CAs) catalyze the hydration and dehydration of carbon dioxide. They are important for regulating ions, fluid and acid-base balance in many tissues. The location of CAs by cell type is important for understanding their roles in these functions. CAs II and VI have been demonstrated using immunohistochemistry (IHC) in the serous acinar cells of human salivary glands and ducts of rat salivary glands. CA IV has been localized by IHC to the ducts of rat salivary glands. CA IV also is present in human parotid glands as shown by real time-polymerase chain reaction (RT-PCR), but this method does not show the distribution of the CA isozymes by cell type. We investigated the cell-specific distribution of CA IV in the human parotid gland. Sections from five formalin fixed, paraffin embedded specimens of human parotid gland were subjected to IHC for CA IV using a commercial antibody. Moderate to strong reactions were found in the cell membranes and cytoplasm of the intercalated, striated and excretory ducts and capillaries, and reactions in the acini were limited to faint areas in some cells. These results indicate that CA IV participates in the regulation of bicarbonate/carbon dioxide fluxes in the ductal system of the human parotid gland.

Carbonic anhydrase (CA) (E.C.4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversable hydration of carbon dioxide (Carpentier et al. 1985; Goto et al. 2008; Ogawa et al. 1998; Parkkila et al. 1990). Functions of CA include macromolecular stabilization, gaseous exchange, acid-base balance, ion transport and bone resorption (Tashian 1989; Carpentier et al. 1985; Ogawa et al. 1998; Parkkila et al. 1990). CA isoenzymes are widely distributed in nearly all mammalian species and approximately 14 isoenzymes have been found in many cell types and tissues including erythrocytes, liver, lung (Zhu and Sly 1990), pancreas, kidney (Spicer et al. 1982) and salivary glands (Parkkila et al. 1990). CAs in salivary glands assist with ion transport, secretory processes and saliva production. In the saliva, CAs are thought to participate in regulation of pH and buffering capacity, which are important for antimicrobial and digestive enzyme activities (Blair-West et al. 1980; Hennigar et al. 1983). Therefore, the location of CAs by cell type has been important for understanding their roles in these functions. For example, isozymes of CA produced and secreted by the acini and located in the cytosol or membranes of striated and excretory ducts are involved in the control of salivary pH via sodium bicarbonate, the principal buffer of stimulated saliva of human and rat major salivary glands (Piras et al. 2012; Ohana 2015). CA isozymes I, II, and VI have been localized immunohistochemically (IHC) in both rodent (Hennigar et al. 1983; Noda et al. 1986a; Ogawa et al. 1998; Peagler et al. 1998; Redman et al. 2000) and human (Noda et al 1986b; Ogawa et al. 1993, Piras et al. 2012; Parkkila et al. 1990) salivary glands; CAs III (Asari et al. 1991) and IV and IX (Cho et al. 2009) are found only in rat salivary glands. CAs IV and VII mRNAs have been demonstrated to be present in human salivary glands by RT-PCR/northern blot (Fujikawa-Adachi et al. 1999), but this method did not show the distribution of the CA isozymes by cell type. We investigated the distribution of CA IV in the human parotid gland using IHC.

Material and methods

Tissue samples

Five neutral buffered formalin fixed, paraffin embedded specimens of normal human parotid gland that had been surgically removed for treatment of salivary gland neoplasms or head and neck cancer were obtained from the archives of the Pathology and Laboratory Service. Permission to use the specimens for research was obtained from the Institutional Review Board Subcommittee and Research and Development Committee of the Washington, DC Veterans Affairs Medical Center.

Histochemistry and immunohistochemistry

Several 4 µm sections were cut from each block, one of which was stained with hematoxylin and eosin (H & E) to ensure that the tissue was suitable for the study. Criteria for suitability included intact, well stained structures indicative of adequate fixation, smooth sections indicative of adequate tissue processing and embedding, and no acinar atrophy or inflammation that would suggest an obstructed duct or an autoimmune disorder or infection.

Other sections were subjected to IHC. Mouse monoclonal anti-human CA IV antibodies [CA IV (E-6): sc-390371] were purchased from Santa Cruz Biotechnology, Inc., Dallas, TX. Immunostaining was localized as a brown precipitate by reaction with H₂O₂ and 3,3 diaminobenzidine (DAB), using Envision™ and dual link HRP in a Dako (Carpenteria, CA) autostainer, and counterstained with hematoxylin. Native peroxidase was quenched with 3% H₂O₂. Reactions were enhanced by antigen retrieval using a citrate Envision™ Flex Target Retrieval solution at pH 6.1 in a 97° C water bath. The antibody dilution and reaction conditions were adjusted for optimal results in a preliminary experiment with human colon and kidney for positive controls. Criteria for optimal results included minimal background staining and epithelial reactions in the control sections that were similar in cellular distribution and intensity to those in illustrations of published reports. For negative controls, the primary antibody was replaced with a negative control cocktail of mouse IgG1, IgG2a, IgG2b, IgG3 and IgM.

Results

Photomicrographs of representative sections of CA IV stained using IHC are presented in Figure 1. Reaction intensity (precipitate density) was moderate to marked in the cytoplasm of all ducts, intercalated, striated and excretory (A–D). In excretory ducts, more staining was observed on the luminal side. No reaction occurred in the acini except for faint patches in scattered cells (A, C). Moderate reactions also were observed in capillaries (B). Reaction was weak to moderate in erythrocytes (D). Reaction in the colon positive control was strong in the luminal membrane and moderate in the cytoplasm of the brush border epithelium of the colon (E) and in some areas, also in the mucous cells. In the kidney positive control, reaction was moderate to strong in the proximal tubules and loops, but there was no reaction in the glomeruli (not illustrated). No reaction occurred in the negative control (F).

Figure 1.

Photomicrographs of immunohistochemically localized CA IV (brown deposits of reaction product) in human parotid glands and colon. A-C) Moderate to strong reactions are in the cytoplasm and luminal membranes of intercalated (i), striated (s) and excretory (e) ducts and capillaries (c). Reaction in the acini (a) was limited to faint patches in some of the cells. D) A mild (center) to moderate (border) reaction occurred in erythrocytes (arrow). E). Colon positive control. A strong reaction is seen in the luminal membrane (arrow) of cells of the brush border and moderate to weak reactions in the cytoplasm of the brush and mucous cells F). Parotid gland negative control. No reaction. Hematoxylin counterstain. Magnification bar = 50 µm (C, D), 100 µm (A, B, E), and 150 µm (F).

Discussion

CA IV is attached to cell membranes of endothelium in lung and in epithelial cells of kidney by a phosphatidylinositol-glycan anchor (Zhu and Sly 1990; Waheed et al. 1992). There they act in concert with various Na⁺, Cl^- and HCO₃^- ion transporters to regulate ion and fluid movement and pH (Alvarez et al. 1999; Schwartz et al. 2000; Ohana 2015). Accordingly, they have been reported to be localized by IHC to the luminal or basolateral membranes of epithelial and capillary endothelial cells of kidney and lung (Brown et al. 1990; Fleming et al. 1995). We found, however, moderate to strong reaction in the cytoplasm of the ducts, which raises concern about the validity of the antibody reaction. Cross-reaction with other CA isozymes is unlikely, because in most reports, CA II and VI reactions have been found in the acini, not the ducts, and CA I does not appear in human salivary glands (Parkkila et al. 1990; Ogawa et al. 1993; Piras et al. 2012). Noda et al. (1986b), however, reported that CAs I and II reacted not only in serous acini but also in the striated and excretory ducts of human salivary glands. In addition, the antibody we used was raised in mice to a specific epitope of human CA IV. Therefore, a more plausible explanation for the reaction in both the plasma membrane and cytoplasm of ducts that we observed is that the antibody recognized CA IV during production and transport within the cytoplasm. This possibility is supported by the reactions of CA IV in both cytoplasm and cell membranes of brush border cells in human colon (Saarnio et al. 1998) and in the luminal cell membrane of ruffle-ended ameloblasts and the cytoplasm of smooth-ended ameloblasts in developing teeth of mouse (Reibring et al. 2014). In addition, the pattern of CA IV reaction in erythrocytes in Figure 1D is like that reported by Wistrand et al. (1999), although some studies have found expression of CAs I, II, III and VI in erythrocytes in salivary glands (Hennigar et al. 1983; Spicer et al. 1990).

It is important to emphasize that the distribution of CA isozyme reactions among salivary gland parenchymal elements differs markedly according to species, source of antibody, fixative, and gland and species tested. Goat and rabbit anti-human erythrocyte CA I do not react at all in rat (Noda et al. 1986a) and human (Parkkila et al. 1990) salivary glands. Rabbit anti-human erythrocyte CA I reacts only with acini in rat parotid and submandibular glands following Carnoy fixation and not at all with Bouin fixation (Hennigar et al. 1983). Sheep anti-human erythrocyte CA I reacts only with ducts in rat salivary glands fixed in Helly’s fluid (Peagler et al. 1998; Redman et al. 2000). Rabbit anti-human erythrocyte CA II reacts strongly with serous acini and weakly in scattered cells of striated ducts in human salivary glands (Parkkila et al. 1990; Ogawa et al 1993). Sheep (Peagler et al. 1998; Redman et al. 2000) and rabbit (Noda et al. 1986a) anti-human erythrocyte CA I and II antibodies react moderately to strongly in intercalated, striated and excretory ducts, but not in acini or erythrocytes, in rat salivary glands fixed in Carnoy’s, Bouin or Helly’s fixatives. Anti-human CA II from other sources reacts only with ducts in rat salivary glands (Ogawa et al. 1998). Anti-horse striated muscle antibodies react with the cytoplasm of striated and excretory ducts in rat parotid gland (Asari et al. 1991) and with myoepithelial cells in human mammary and prostate glands (Väänänen and Autio-Harmainen 1987). Anti-human and anti-rat salivary CA VI antibodies react only with serous acini, including the secretory granules, of human salivary glands (Ogawa et al. 1993, 1998; Parkkila et al. 1990; Piras et al. 2012) and only in ducts of rat salivary glands (Peagler et al. 1998; Redman et al. 2000). Our findings allow anti-human CA IV reactions in human parotid gland ducts to be added to this list.

Our results indicate that CA IV participates in regulating bicarbonate/carbon dioxide flux in the ductal system of the human parotid gland. We propose that CA IV may play an important role in regulating salivary pH, which is important for antimicrobial and digestive enzyme activities.